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目的 基于抑制补体活化的两个关键环节,即C3/C5转化酶及MAC的形成,设计、构建及制备一种新型、高效的补体抑制剂。方法 首先设计引物,通过PCR技术扩增重组可溶性CD46/CD55/CD59嵌合分子cDNA片段,重组于pcDNA3.1(+)真核表达载体上,构建兼有三分子功能的CD46/CD55/CD59嵌合补体抑制分子,命名为HCI-3(Human Chimeric Complement Inhibitor,HCI-3)。分别利用COS-7细胞和CHO细胞进行表达,并用抗人CD46多抗及抗人CD55,CD59单抗对表达产物进行Western Blotting鉴定。结果 DNA测序结果证实,前端带有CD59信号肽序列,后端融合有编码6个组氨酸碱基的HCI-3 cDNA片段的阅读框完整。免疫印迹结果显示,表达的重组蛋白分别能与抗人CD46多克隆抗体和抗人CD55,CD59单抗结合。结论 成功构建并在真核细胞内表达了重组可溶性HCI-3分子,为进一步研制和开发新一代多功能、多靶点补体抑制剂奠定了基础。
OBJECTIVE To design, construct and prepare a novel and efficient complement inhibitor based on the formation of C3 / C5 convertase and MAC based on two key components of inhibiting complement activation. Methods Primers were designed. The cDNA fragments of recombinant soluble CD46 / CD55 / CD59 chimeric molecules were amplified by PCR and cloned into pcDNA3.1 (+) eukaryotic expression vector to construct a three-molecule CD46 / CD55 / CD59 chimeric Complement inhibitory molecule, named HCI-3 (Human Chimeric Complement Inhibitor, HCI-3). The expressed products were identified by Western Blotting using COS-7 cells and CHO cells, respectively, and anti-human CD46 polyclonal antibody and anti-human CD55, CD59 monoclonal antibody. Results The results of DNA sequencing confirmed that the reading frame of the HCI-3 cDNA fragment with the CD59 signal peptide at the front end fused with 6 histidine bases was intact. Western blotting results showed that the expressed recombinant protein could bind to anti-human CD46 polyclonal antibody and anti-human CD55 and CD59 monoclonal antibody, respectively. Conclusion The successful construction and expression of recombinant soluble HCI-3 molecules in eukaryotic cells lay the foundation for the further development and development of a new generation of multifunctional and multi-target complement inhibitors.